RESUMO
The toxicity of fatty acid salts to German, Blattella germanica (L.), and American cockroaches, Periplaneta americana (L.), was evaluated. Potassium and sodium laurate caused up to 95% mortality of German cockroaches and 100% mortality of American cockroaches. Even-numbered potassium fatty acid salts, C8-C18 were assessed for toxicity at 0.125, 0.25, 0.5, 1, and 2% concentrations by a 30-s immersion of cockroaches. The more soluble of the fatty acid salts at 2% concentration caused 65-95% mortality of German cockroaches and 100% mortality of American cockroaches. Potassium oleate, C18, was most toxic to both German (LC50 = 0.36%) and American (LC50 = 0.17%) cockroaches. Fatty acid salt solutions on a substrate were tested by placing cockroaches in contact with treated floor tiles immediately after application (wet) or after the solutions had dried. Sodium laurate and potassium caprate caused mortality of German (62 +/- 17.4 and 58 +/- 12.6%, respectively) and American cockroaches (52 +/- 18.5 and 28 +/- 4.9%, respectively) on wet tiles, whereas potassium oleate caused mortality of German cockroaches (67 +/- 14.1%) only. Dry fatty acids caused no mortality among exposed cockroaches. Fatty acid salt solutions can be effective in killing German and American cockroaches but only when insects are thoroughly wetted with 1-2% fatty acid salt solutions.
Assuntos
Baratas , Sabões , Animais , Masculino , Sabões/química , Relação Estrutura-AtividadeRESUMO
Monoclonal antibodies (MAbs) that are candidates for antibody-directed therapy were evaluated by a flow cytometric method. This method accurately quantitates the intensity of staining and the percentage of cells from freshly derived primary tumors expressing the relevant cell surface antigens. This method was applied to human colorectal, gastric, and ovarian carcinomas. It allowed calculations of the number of drug molecules that potentially could be delivered by each MAb as well as selection of the optimal combinations of antibodies for treatment of each type of cancer. The binding of all the MAbs varied among the tumors, although combinations of antibodies reduced this problem. A combination of MAbs C14 and NCRC-23 recognized 97% of colorectal tumors. A combination of C14, NCRC-23, and 791T/36 recognized 95% of gastric tumors. Combinations of either 791T/36 and C14 or 791T/36 and NCRC-11 recognized 80% of ovarian tumors. The number of cells binding with a single MAb varied within the tumor. The optimal anti-colorectal tumor antibody was NCRC-23 (anti-carcinoembryonic antigen), which recognized a mean of 65% of the large cells within a tumor at a mean antigen density of 4.9 X 10(5) sites/cell. The optimal anti-gastric tumor antibody was C14 (anti-Y hapten), which recognized a mean of 66% of the large cells within a tumor at a mean antigen density of 4.4 X 10(5) sites/cell. The optimal anti-ovarian antibody was 115/D8, which recognized 54% of the large cells at a mean antigen density of 4.2 X 10(5) sites/cell. These antigen densities were similar to those calculated for HLA/ABC antigens in colorectal and ovarian cancers. However, the gastric tumors expressed elevated levels of major histocompatibility complex class I antigens, with a mean density of 7.3 X 10(5) sites/cell. Combinations of antibodies that recognize a high proportion of tumor cells are likely to be necessary for MAb-drug targeting to prevent tumor recurrence and/or metastases.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Antígenos de Neoplasias/análise , Antígenos de Superfície/análise , Citometria de Fluxo , Imunotoxinas/farmacologia , Neoplasias/imunologia , Imunofluorescência , Antígenos HLA/análise , Humanos , Fenótipo , Coloração e RotulagemRESUMO
Ten preparations of BCG, six clinical vaccines, and four experimental preparations were compared for suppression of tumor growth by regional application. The preparations differed widely in their proportions of viable bacterial units and in bacterial unit:dry weight ratios. As assessed by their ability to suppress tumor development following direct admixtures with cell inocula of a rat sarcoma, one of the six clinical vaccines (Connaught) was significantly superior to Glaxo on any parameter (dry weight, No. of total units, or No. of viable units), immuno BCG Pasteur F was superior to Glaxo on two parameters (dry weight and No. of viable units), and Pasteur scarification was superior to Glaxo only on a viable unit basis. The Tice and Rijks Institute vaccines were not significantly different from Glaxo on any basis. Experimental vaccines from the Trudeau Mycobacterial Collection, stored as frozen liquid suspensions, showed a less marked variation in physical properties; here too, the Pasteur strain was superior to two other Trudeau preparations examined (Tice and Phipps). Viable organisms were not essential for tumor suppression, gamma-radiation-sterilized vaccine being equally effective. Tests with pulmonary tumor deposits, treated by iv BCG, and tests with pleural deposits, treated by intrapleural BCG, indicated that agents identified as superior in the subcutaneous screening system were also superior in the treatment of thoracic deposits.
Assuntos
Vacina BCG/uso terapêutico , Sarcoma Experimental/terapia , Animais , Vacina BCG/administração & dosagem , Vacina BCG/isolamento & purificação , Injeções Subcutâneas , Neoplasias Pulmonares/terapia , Masculino , Neoplasias Pleurais/terapia , Ratos , Neoplasias de Tecidos Moles/terapiaRESUMO
There is considerable interest in the development of anti-idiotypic antibodies as vaccines in a number of diseases, including cancer. We have developed a human anti-idiotypic monoclonal antibody (105AD7) which binds at or very near to the binding site of mouse antitumor monoclonal antibody 791T/36. The 791T/36 antibody binds to a tumor-associated antigen (gp72) expressed on a number of human tumors, including colorectal and ovarian carcinomas and osteogenic sarcoma. This study shows that, in rats and mice, 105AD7 induces delayed-type hypersensitivity to human tumor cells bearing the gp72 antigen. Local transfer of delayed hypersensitivity was also demonstrated using lymphocytes from mice primed with 105AD7. These findings show that the human monoclonal anti-idiotypic antibody 105AD7 is likely to induce cellular immune responses to tumors in cancer patients.
Assuntos
Anticorpos Anti-Idiotípicos/farmacologia , Anticorpos Monoclonais/farmacologia , Hipersensibilidade Tardia/imunologia , Imunidade Celular/efeitos dos fármacos , Neoplasias Experimentais/imunologia , Animais , Feminino , Humanos , Imunoglobulina G/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Ratos Endogâmicos , Células Tumorais CultivadasRESUMO
Improvements have been made in the synthesis of a drug-carrier-antibody conjugate using methotrexate as the drug, human serum albumin as the carrier, and a monoclonal antibody against a human osteogenic sarcoma cell line (791T/36). The improvements have resulted in a higher and more reproducible substitution of serum albumin by methotrexate, and improvements in the coupling of methotrexate covalently linked to human serum albumin to antibody resulting in a greater ease and efficiency of conjugation. The improvements have led to a conjugate of increased cytotoxicity while retaining the previously reported specificity. A conjugate is reported which shows cytotoxicity of 1.1 ng/ml (2.4 nM) with respect to methotrexate and 6 X 10(-11) M with respect to antibody in a clonogenic assay on 791T cells. This cytotoxicity is greater than that obtained using free methotrexate (2.8 ng/ml; 6.1 nM) and implies that drug cytotoxicity can be considered as the sum of drug uptake and the number of drug molecules required to kill a cell. This further suggests that antibodies could provide a potent delivery system for drugs which are poorly taken up by cells.
Assuntos
Metotrexato , Osteossarcoma/imunologia , Anticorpos Monoclonais/uso terapêutico , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Concentração de Íons de Hidrogênio , Imunoquímica , Metotrexato/administração & dosagem , Osteossarcoma/tratamento farmacológico , Selenometionina/metabolismo , Albumina SéricaRESUMO
Fab/c fragments, purified from pepsin digest of mouse IgG2b monoclonal antibody (MoAb) 791T/36, consist of one Fab arm and the intact Fc portion. Pharmacokinetic and biodistribution studies in BALB/c mice of radioiodine-labeled 791T/36 MoAb and its Fab/c fragments showed that, due to the presence of the Fc portion, the Fab/c fragment has the same catabolic rate as whole antibody (T1/2 = 64 h). Due to its lower molecular weight (105,000), the Fab/c fragment extravasated more quickly and to a greater extent than whole MoAb in organs in which the vascular endothelium was fenestrated or continuous. In organs in which the vascular endothelium is sinusoidal, such as in liver and spleen, their diffusion capacities were identical. Therefore, Fab/c fragments reconcile advantages of the intact antibody molecule (slow catabolic rate) and Fab or F(ab)2 fragments (increased extravascular diffusion), features required to improve targeting to solid tumors. Data from biodistribution studies in nude mice bearing subcutaneous 791T tumor (antigen positive) and Colo205 tumor (antigen negative) contralaterally showed important differences in the behavior of whole MoAb and Fab/c fragment: (a) Whole MoAb was cleared more rapidly from the body and from the blood than Fab/c fragment; (b) The MoAb was taken up by the spleen (tissue to blood ratio greater than 1 from 12 h after injection over the 3 days of the experiment) and the liver (0.6), whereas Fab/c fragment tissue to blood ratios were only slightly increased (0.34 and 0.35) compared to control nude mice (0.25 and 0.28) for the spleen and liver, respectively, 3 days after injection. Since both MoAb and Fab/c fragment bear the Fc portion, these data suggest that the reputed "nonspecific uptake" of antibodies due to the Fc portion could be an Fc-mediated specific uptake, e.g., uptake of immune complexes; (c) The tumor to blood ratios were 1.7 and 1.2 for MoAb and Fab/c fragment, respectively, from 24 h throughout the experiment, whereas the percentage of injected dose (% of ID) present/g of 791T tumor was at any time greater for Fab/c fragment (8% maximum of ID) than for MoAb (5% of ID). These results were not expected in view of the low immunoreactivity in vitro of Fab/c fragments compared to whole antibody. It is suggested that the distribution and the catabolic rate of whole antibody and its Fab/c fragment at the tumor level are modulated by their respective valency and immunoreactivity for the target cell.
Assuntos
Anticorpos Monoclonais/farmacocinética , Fragmentos de Imunoglobulinas/farmacocinética , Animais , Neoplasias do Colo/imunologia , Humanos , Taxa de Depuração Metabólica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de NeoplasiasRESUMO
Immunotoxin constructed by conjugating ricin A chain to monoclonal antibody 791T/36 specifically inhibits growth of human tumor xenografts which express the gp72 antigen recognized by the antibody component. Dose schedule tests showed that the major response was obtained during the first 5 days of treatment and further prolonged treatment did not improve therapy. Expression of gp72 antigen on tumor cells derived from xenografts in immunotoxin-treated mice was not markedly altered indicating that treatment did not lead to the expansion of tumor antigen deficient tumor cells. The experiments indicate that treatment for short duration with immunotoxin may be the most effective protocol.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Imunotoxinas/uso terapêutico , Neoplasias Experimentais/terapia , Ricina/uso terapêutico , Animais , Antígenos de Neoplasias/análise , Humanos , Imunotoxinas/imunologia , Imunotoxinas/toxicidade , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Osteossarcoma/terapia , Transplante HeterólogoRESUMO
Fifty colorectal tumors were screened by indirect immunofluorescence and flow cytometry for antigen expression using a panel of monoclonal antibodies that recognize determinants preferentially expressed on tumor cells (carcinoembryonic antigen, Y haptenic blood group, 791T/36 defined antigen 791T-P72). Fifty % of the tumors expressed all three antigens, 41%, two, and 9%, one. Over a third reacted strongly with at least one monoclonal antibody, although the majority of tumors stained with a moderate intensity. Extranuclear membranes from tumors showed similar antigen expression to disaggregated tumor cells and were particularly useful for providing the relative tumor:normal tissue binding ratios. The carcinoembryonic antigen specific monoclonal antibody showed the strongest tumor selectivity with a tumor:normal tissue ratio of 24 +/- 7:1. Lack of correlation between expression of the three antigens suggested that the monoclonal antibodies recognizing them may have potential as a "cocktail." One-third of the tumors contained cells with an aneuploid DNA content and expressed elevated levels of carcinoembryonic antigen and Y haptenic blood group antigen when compared to tumors with diploid DNA content. Aneuploid cells within a tumor were also preferentially stained with all of the monoclonal antibodies.
Assuntos
Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Antígenos de Neoplasias/análise , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , DNA de Neoplasias/análise , Neoplasias Retais/imunologia , Neoplasias Retais/patologia , Anticorpos Monoclonais , Citometria de Fluxo , Imunofluorescência , Humanos , Membranas/imunologiaRESUMO
Ricin toxin A chain (RTA) was conjugated to monoclonal antibody 791T/36, which was raised originally against human osteogenic sarcoma cell line 791T. The resultant conjugates were characterized and tested for cytotoxicity against a panel of human tumor cell lines representing a defined range of antigenicity with regard to 791T/36. Conjugates were highly cytotoxic for cells expressing high antigen density, inhibiting cell survival at RTA concentrations three to four orders of magnitude lower than that possible with RTA alone. Cytotoxicity of conjugates diminished with decreasing 791T/36 antigen concentration on target cells, but significant effects were seen against cells of low or intermediate antigenicity. Cytotoxicity could be blocked specifically by excess 791T/36 antibody, clearly indicating that antigen binding was a necessary part of the mechanism of action. Comparison with drug-antibody conjugates indicated that RTA immunotoxins are much more active, but discriminate less readily than drug-antibody conjugates between cells of different antigenicity. It is suggested that these properties be taken into account with regard to practical application and future development.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Antineoplásicos/administração & dosagem , Imunotoxinas/toxicidade , Ricina/administração & dosagem , Especificidade de Anticorpos , Ligação Competitiva , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Metotrexato/administração & dosagem , Osteossarcoma/imunologia , Fatores de TempoRESUMO
The efficacy of monoclonal antibody therapy depends in part on the expression of the relevant tumor-associated antigens by both primary tumors and their metastases. Antigen expression by paired primary and autologous metastases from surgically excised osteogenic and soft-tissue sarcomas from 15 patients was studied using a panel of murine hybridoma monoclonal antibodies and indirect immunoperoxidase staining of formalin-fixed tissue sections. The panel included three antibodies (B3619, 17-9H3, OST6) recognizing sarcoma-associated antigens and an antibody recognizing an HLA-DR framework determinant (OKla1). In most cases, antibody binding to both primary and metastatic tumors was observed. However, marked heterogeneity of binding intensity between primary and metastatic tumors and of cells expressing antigens within tumors was noted. This occurred even though primary and metastatic tumors demonstrated homogeneous histology and cellular morphology. Differences were noted among patients as well as among metastases taken from an individual. A significant number of both primary and metastatic tumors contained cells that did not bind a particular antibody even in the presence of other cells that demonstrated significant antibody binding. Thus, strategies for single monoclonal antibody therapy may be limited by heterogeneity of intertumor and intratumor antigenic expression.
Assuntos
Antígenos de Neoplasias/análise , Sarcoma/imunologia , Animais , Anticorpos Monoclonais , Complexo Antígeno-Anticorpo , Fibrossarcoma/imunologia , Humanos , Técnicas Imunoenzimáticas , Camundongos , Metástase Neoplásica , Osteossarcoma/imunologia , Sarcoma/patologia , Neoplasias de Tecidos Moles/imunologiaRESUMO
A series of four monoclonal antibodies was raised against suspensions of normal adult WAB/Not rat hepatocytes. An immunoperoxidase-staining technique was used to examine the distribution of determinants detected by these antibodies on frozen sections of fetal, neonatal, adult, and regenerating liver and on a range of 4-dimethylaminoazobenzene-induced liver lesions, including a panel of 32 primary liver carcinomas. Three of the antibodies were directed against hepatocytes, while a fourth antibody stained stromal elements within the liver. The determinants detected by the anti-hepatocyte monoclonal antibodies arose in a specific sequence during normal liver development and, when assessed in conjunction, characterized several phenotypes associated with stages in normal hepatocyte differentiation. These same antibody-defined phenotypes were expressed by the primary liver carcinomas, and the distribution of phenotypes among the tumors revealed a heterogeneity which was not evident from a conventional morphological classification. Primary liver tumors expressed a total of four antibody-defined phenotypes, whereas gamma-glutamyl transpeptidase-positive foci of hepatocytes and neoplastic nodules expressed, respectively, only one or 2 antibody-defined phenotypes. We suggest that monoclonal antibodies directed against normal liver cell components may provide a means to establish lineage relationships between cell populations involved in hepatocarcinogenesis.
Assuntos
Anticorpos Monoclonais , Neoplasias Hepáticas Experimentais/imunologia , Neoplasias Hepáticas/imunologia , Fígado/imunologia , Animais , Complexo Antígeno-Anticorpo , Antígenos de Neoplasias/análise , Antígenos de Superfície/análise , Epitopos/análise , Técnicas Imunoenzimáticas , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/patologia , Regeneração Hepática , Ratos , Ratos Endogâmicos , p-DimetilaminoazobenzenoRESUMO
Immunotoxin constructed by conjugating ricin A chain to monoclonal antibody 791T/36 has a markedly altered biodistribution when compared to unconjugated antibody. This is principally manifest as hepatic uptake of immunotoxin which appears to be controlled by the ricin A chain (RTA) moiety. This was established by comparing the blood survival and organ distribution of immunotoxin with that of ricin A chain and free antibody using preparations in which either the RTA or antibody, alone or as components of the immunotoxin, was radiolabeled. Gel filtration chromatography of sera from immunotoxin treated animals demonstrated a preferential blood clearance of immunotoxin with high RTA-antibody ratio. Hepatic uptake is dependent upon Kupffer cell recognition of mannose-containing oligosaccharide structures on the RTA moiety of immunotoxin. Mannose-containing blocking agents given with immunotoxin were shown to prolong circulation time of the immunotoxin in blood including those species with higher RTA-monoclonal antibody ratios and reduce liver uptake. Effective blocking agents include ovalbumin, ovomucoid, and mannosyl-lysine (Man3Ly2). These studies demonstrate that agents specifically inhibiting hepatic uptake of immunotoxin significantly alter biodistribution and may improve their therapeutic efficacy.
Assuntos
Anticorpos Monoclonais , Imunotoxinas/farmacocinética , Fígado/metabolismo , Ricina/farmacocinética , Animais , Meia-Vida , Células de Kupffer/metabolismo , Mananas/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/farmacologia , Ovomucina/farmacologia , Distribuição TecidualRESUMO
Monoclonal antibody 791T/36, recognizing a Mr 72,000 antigen on the surface of colon carcinoma cells, has been used to construct an immunotoxin by conjugating to it the ribosomal inhibitor protein, ricin toxin A chain. The antibody 791T/36 has been shown to bind to membranes of freshly disaggregated tumor cells from human colon tumors, and to localize in tumors in vivo. Subacute toxicology testing in rats receiving immunotoxin i.v. showed, at highest doses, weight loss, decreased serum albumin, and hepatocyte vacuolization without elevation in liver function tests. A Phase I dose escalation study was carried out in which 17 patients with metastatic colorectal cancer were treated with doses of immunotoxin ranging from 0.02 to 0.2 mg/kg/day in 1-h i.v. infusions for a 5-day course. Side-effects included a composite of signs and symptoms thought to be generic to ricin A chain immunotoxins, including decreased serum albumin, mild fever, and flu-like symptoms, all being reversible. Two additional findings, reversible proteinuria and mental status changes, were also noted which may be characteristic of this immunotoxin. By 10-20 days after therapy, most patients developed IgM and IgG antibodies against both the ricin toxin A chain and the immunoglobulin portion of the immunotoxin, which were asymptomatic. A strong anticombining site antibody response was seen. Biological activity manifest as mixed tumor regression was seen in five patients.
Assuntos
Anticorpos Monoclonais/efeitos adversos , Neoplasias do Colo/terapia , Imunotoxinas/efeitos adversos , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/secundário , Ricina/efeitos adversos , Adulto , Idoso , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais/toxicidade , Formação de Anticorpos , Antígeno Carcinoembrionário/análise , Neoplasias do Colo/imunologia , Avaliação de Medicamentos , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/análise , Imunoglobulina M/análise , Imunotoxinas/uso terapêutico , Imunotoxinas/toxicidade , Dose Letal Mediana , Testes de Função Hepática , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/terapia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/terapia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Metástase Neoplásica , Ratos , Ratos Endogâmicos , Ricina/uso terapêutico , Ricina/toxicidade , Albumina Sérica/análiseRESUMO
The immunogenicity of human anti-idiotypic antibody has been investigated using a human monoclonal anti-idiotypic antibody (105AD7) which interacts with the binding site of 791T/36, a mouse monoclonal antibody against gp72 antigen. This antigen is frequently expressed in gastrointestinal cancer, therefore, six patients with advanced colorectal cancer have been immunized with 105AD7 as an aluminum hydroxide gel precipitate in a phase I clinical study. Cryopreserved blood mononuclear cells were tested for in vitro proliferative responses by [3H]thymidine incorporation; plasma samples were tested by enzyme-linked immunosorbent assay for anti-anti-idiotypic and antitumor antibodies, and for interleukin 2. Proliferative responses to gp72 positive tumor cells were seen in four of five patients tested; parallel in vitro responses to 105AD7 anti-idiotypic antibody were seen in most of these patients. Interleukin 2 was detected in the plasma of four of six patients after 105AD7 immunization, with peak levels up to 7 units/ml. No toxicity related to anti-idiotype immunization and no antitumor or anti-anti-idiotype antibodies were seen. This study shows that human monoclonal anti-idiotype 105AD7 is immunogenic in cancer patients, inducing cellular antitumor responses and interleukin 2 production. This suggests that human monoclonal anti-idiotype antibodies may have considerable potential for immunotherapy of human cancer.
Assuntos
Anticorpos Anti-Idiotípicos/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Neoplasias Colorretais/terapia , Imunoterapia Ativa , Interleucina-2/sangue , Adulto , Idoso , Análise de Variância , Anticorpos Anti-Idiotípicos/efeitos adversos , Anticorpos Monoclonais/efeitos adversos , Divisão Celular/efeitos dos fármacos , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Neoplasias Hepáticas/secundário , Masculino , Pessoa de Meia-Idade , Fatores de TempoRESUMO
Ricin A chain immunotoxin constructed with monoclonal antibody 791T/36, which recognizes a tumor associated glycoprotein Mr 72,000 antigen present on sarcomas and colon and ovarian cancer cells, is cytotoxic for cell lines from tumors expressing this antigen. Incubation of sarcoma 791T cells with immunotoxin for only 5 min is sufficient to produce greater than 95% inhibition of tumor cell growth. Papain treatment of these cells to remove immunotoxin from the cell surface indicated that the cell surface acts as a reservoir for continued internalization of immunotoxin over several hours, but even so, 50% inhibition of cell survival was produced over a 2- to 3-h period. Analysis of the rate of endocytosis demonstrated that 30-50% of cell bound immunotoxin was internalized over a 180-min period. This was primarily dictated by the antibody moiety, regardless of the degree of conjugation to ricin A chain. This rate is much slower than that of other cell surface ligands such as transferrin. Cell cytosol acidification experiments were performed to determine whether this immunotoxin was internalized by clathrin coated pits, which is relatively rapid, or by smooth pits, which is slower, and the results indicated the latter mechanism is almost exclusively used. Intracellular trafficking of antibody 791T/36, conjugated to human serum albumin-tetramethylrhodamine was investigated by flow cytometry. The movement of the conjugate into the lysosomal compartment was delayed so that degradation products were only detected after a lag phase of 30-60 min. The lack of potentiator dependence of 791T/36 immunotoxin is in keeping with these findings.
Assuntos
Anticorpos Monoclonais/farmacocinética , Endocitose , Imunotoxinas/farmacocinética , Lisossomos/metabolismo , Ricina/farmacocinética , Ensaio Tumoral de Célula-Tronco , Cloreto de Amônio/farmacologia , Neoplasias do Colo/metabolismo , Feminino , Humanos , Monensin/farmacologia , Neoplasias Ovarianas/metabolismo , Sarcoma/metabolismo , Fatores de Tempo , Células Tumorais Cultivadas/metabolismoRESUMO
Flow cytofluorimetric methods have been used to quantitate the interaction between a divalent monoclonal antibody and a tumour cell surface antigen. After standardization using fluorescein and 125I-labelled antibodies, kinetics of association and dissociation were measured, and antibody bound at equilibrium quantitated. A mathematical model was developed in conjunction with these experimental results which allowed the calculation of rates for monovalent association and monovalent and divalent dissociation, and a description of the contribution of each to the level of bound antibody at different antibody concns.
Assuntos
Anticorpos Monoclonais/imunologia , Reações Antígeno-Anticorpo , Antígenos de Neoplasias/imunologia , Antígenos de Superfície/imunologia , Osteossarcoma/imunologia , Linhagem Celular , Citometria de Fluxo , Humanos , Cinética , Modelos BiológicosRESUMO
The protein core of high mol. wt polymorphic epithelial mucin (PEM--approximately 400 kDa glycoprotein) which is associated with breast carcinomas, consists of a repeating 20 amino acid peptide motif [Gendler et al. (1988) J. biol. Chem. 263, 12,820-12,823]. Monoclonal antibodies C595 (anti-urinary mucin) and NCRC-11 (anti-breast carcinoma cells), and other antibodies against human milk fat globule membranes, were found to recognize determinants present within this 20 amino acid peptide. A model of the peptide was developed based on hydropathicity and structure prediction calculations and these indicated that the repeated structure is dominated by a hydrophilic domain of seven amino acids, extending into two flanking beta turns. NMR analysis of the 20 amino acid peptide was undertaken to probe the secondary structure. Epitope mapping experiments involving solid phase synthesis of overlapping heptapeptides in the repeat unit identified the minimum structures for antibody binding as Arg-Pro-Ala-Pro and Arg-Pro-Ala for the C595 and NCRC-11 antibodies, respectively. These determinants were found within the predicted hydrophilic turn region domain of the peptide. The epitopes for six other PEM-reactive monoclonal antibodies were also determined to reside within the predicted hydrophilic turn domain. This evidence is in accord with the disposition of this region of the PEM peptide core being at the exterior of the glycoprotein where it would be accessible to antibody recognition and binding events.
Assuntos
Glicoproteínas de Membrana/imunologia , Mucinas/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais , Neoplasias da Mama/imunologia , Reagentes de Ligações Cruzadas , Epitopos/imunologia , Humanos , Espectroscopia de Ressonância Magnética , Glicoproteínas de Membrana/urina , Dados de Sequência Molecular , Mucina-1 , Mucinas/urina , Peptídeos/síntese química , Peptídeos/imunologia , Conformação Proteica , Soroalbumina BovinaRESUMO
Trichosanthin, a ribosomal inhibitor protein, blocks HIV replication in lymphocytes and macrophages. This agent was used to treat 51 patients with advanced HIV disease in a dose-escalation study in which three injections were administered over a 9-21-day period in a dose range of 10-30 micrograms/kg per injection. The maximum tolerated dose was estimated to be 30 micrograms/kg. Reversible but severe fatigue and myalgias were the major dose-limiting side-effects; mild leucocytosis and elevations in serum transaminases were noted and were reversible. Non-dose-related reversible mental status changes were seen in six patients and were considered to be associated with the drug. This was usually manifest as dementia, but progressed to coma in two patients. This reversed, but the sequelae resulted in death in one patient. Decreases in serum p24 antigen levels were noted 1 month after the first infusion in 10 of 18 patients who entered the study with elevated levels; one converted to negative. Values usually remained low to the end of the study period (2 months). In those patients with CD4+ cell levels greater than 50 x 10(6) cells/l significant decreases in sedimentation rate and increases in CD4+ cell numbers were also noted. These changes were found at all dose levels but only in patients receiving three infusions.
Assuntos
Infecções por HIV/tratamento farmacológico , Tricosantina/uso terapêutico , Adulto , Animais , Anticorpos/sangue , Peso Corporal , Demência/induzido quimicamente , Relação Dose-Resposta a Droga , Avaliação de Medicamentos , Produtos do Gene gag/sangue , Antígenos HIV/sangue , Proteína do Núcleo p24 do HIV , Infecções por HIV/imunologia , Humanos , Masculino , Camundongos , Subpopulações de Linfócitos T , Tricosantina/administração & dosagem , Tricosantina/efeitos adversos , Tricosantina/imunologia , Proteínas do Core Viral/sangueRESUMO
The endocytosis of a monoclonal antibody recognising a cell surface glycoprotein antigen has been investigated using several different fluorescent conjugates. These conjugates have been employed for both fluorescence microscopy to show the qualitative changes in distribution of antibody conjugates during endocytosis, and also flow cytofluorimetry to show the quantitative changes in fluorescence intensity associated with this redistribution. Using an antibody directly labelled with fluorescein it was difficult to demonstrate endocytosis due to the inability to distinguish clearly between internal and external fluorescence. However, a fluorescein-HSA-antibody conjugate which was heavily quenched at the cell surface was endocytosed and degraded during incubation at 37 degrees C for 4 h and was then visualised in a perinuclear distribution by the addition of agents to modify intracellular pH. This experiment demonstrated that such conjugates became localised within an acidic internal compartment. A tetramethylrhodamine-HSA-conjugate also demonstrated a similar perinuclear distribution but without the addition of endosomal pH modifiers. When used in conjunction with a fluorescein rabbit anti-HSA second label this conjugate also showed that not all conjugate was endocytosed during a 4-h period; some conjugate remained bound to the cell surface. These experiments suggested that endocytosis in this system differs from receptor-mediated endocytosis via coated pits which is reported to be more rapid and complete.
Assuntos
Anticorpos Monoclonais , Antígenos de Superfície/análise , Endocitose , Glicoproteínas/análise , Linhagem Celular , Citometria de Fluxo , Fluoresceína-5-Isotiocianato , Fluoresceínas , Imunofluorescência , Proteínas de Membrana/análise , Osteossarcoma , TiocianatosRESUMO
Serum IgG immunoglobulin fractions from human subjects hyposensitized to poison ivy/oak by oral administration of urushiol suppressed the induction of delayed-type hypersensitivity (DTH) responses in mice to this hapten. This suppressive activity was hapten specific because it did not modify DTH responses to dinitrofluorobenzene (DNFB). Absorption of human serum with lymph node cells from urushiolsensitized but not DNFB-sensitized mice removed the suppressive activity, suggesting that anti-idiotypic antibodies reacting with T-cell receptors are involved.